Histopathological examination and scoring
Foot pads of the left and right hind paws were isolated on day 25, fixed in 10% neutral buffered
formalin, decalcified with 5% formic acid-formalin, embedded in paraffin and sectioned. The sections
were stained with hematoxylin-eosin and observed under a light microscope.
The area surrounding the 5 metatarsal bones were evaluated and scored based on the degree of
periosteal new bone formation, periostitis, inflammatory cell infiltration and edema. The degree of
periosteal new bone formation and periostitis were designated a score of 0–4 with score 0 of no change,
score 4 of strong changes in more than 4 metatarsal bones. The degree of inflammatory cell infiltration
was designated a score of 0–3 with score 0 of no change, score 3 of changes around more than 3
metatarsal bones measured more than 50 (40 Am scale) in width. A score of 1 was added in the case of
strong focal infiltration. The degree of edema was designated a score of 0–4 with score 0 of no change,
score 4 of changes around more than 3 metatarsal bones measured more than 50 (40 Am scale) in
width. The microscopic score and the paw volume were carried out by an investigator not blinded by the
treatment regime.